Methods and devices to detect target analytes in samples, particularly biological samples, have taken advantage of the specific interactions between biological molecules, such as the interactions between antigen-antibody (or part of antibody, e.g., Fv), ligand-receptor, enzyme-substrate, binding protein-nucleic acid or aptamer, and hybridization of nucleic acid molecules. These interactions allow for the analyte to be isolated or captured from other components in the sample.
A common type of device that incorporates the use of such biological interactions is a lateral flow assay device or strip assay device. Such assay devices typically comprise a reagent pad that contains a binding partner for the analyte of interest coupled to a detectable label (i.e. labeled conjugates) and a porous membrane on which a capture protein (e.g. antibody or antigen) capable of binding the analyte of interest is immobilized. Labeled conjugates that are commonly used in these types of assay devices are antibodies or antigens coupled to gold nanoparticles or colored latex particles. A liquid sample applied to the device travels by capillary action through the reagent pad where any analyte present in the sample binds to the labeled conjugate forming a complex. The complex continues to migrate through the porous membrane to the region where the capture protein is immobilized at which point the complex of analyte and labeled conjugate will bind to the capture protein. The unreacted sample passes through the immobilized capture protein region without binding. The presence of the analyte is then determined by detecting the labeled conjugate in the capture region of the device (e.g. by a color change). Although these devices are amenable for use as rapid clinical tests for some analytes, the nature of these assay devices prevents single-step amplification of the detection signal, thus making such devices inadequate for the detection of a number of analytes that require greater sensitivity.
One method that has been previously employed to enhance the sensitivity of assays that utilize specific biological interactions for the detection of analytes in samples is the use of enzyme labels. The enzyme-linked immunosorbent assay (ELISA) format exemplifies such a method. In these assays, an antibody or antigen capable of binding an analyte of interest is immobilized on the bottom of a microplate well. Sample is added to the well followed by one or more wash steps. A second antibody or antigen coupled to an enzyme is subsequently added to the well. After additional wash steps, a substrate that can be converted by the enzyme into a chromogenic or fluorescent substance is added to the well, and the presence of analyte is determined by detecting the chromogenic or fluorescent substance. Enzyme-labeled conjugates have been incorporated in lateral flow assays to improve their sensitivity. However, such assays require additional steps to add enzyme substrate or wash away unreacted conjugates. Consequently these assays require longer time than lateral flow assays.
Thus, there is a need in the art for additional lateral flow assay devices, e.g., devices that offer greater detection sensitivity. The development of one-step, e.g., rapid lateral flow assay devices with built-in amplification is desirable.